转录因子Tbx18导入人诱导多潜能干细胞分化的心肌细胞构建起搏样细胞的实验研究

Direct conversion of human induced pluripotent stem cell-derived cardiomyocytes to pacemaker cells by expression of transcript factor Tbx18

目的:研究转录因子Tbx18能否转染人诱导多潜能干细胞分化的心肌细胞(hiPSC-CMs)并使其向起搏细胞分化。方法:纯化接种hiPSC-CMs,构建重组腺病毒-绿色荧光蛋白Ad-(GFP)-Tbx18载体,转染hiPSC-Cs为实验组,转染只含GFP的腺病毒组及空白未转染组作为对照。转染后定期检测细胞的变化情况,荧光倒置显微镜计数转染后细胞的搏动频率变化情况;RT-PCR测定起搏细胞特异性基因的表达。结果:实验组hiPSC-CM的搏动频率高于对照组[(61.3±13.6)vs.(33.0±1.67)次/min,P<0.05)]。实验组起搏特异性基因HCN4的表达较对照组明显增加,KIR2.1、SCN5A、CX43等心室肌特异性基因表达较对照组下降,差异有统计学意义(P<0.05)。结论:Tbx18基因可成功转染hiPSCCMs,并使之转化为窦房结样起搏细胞。

Objective： To observe if Tbx18 can successfully transfer human induced pluripotent stem cell-derived cardiomyocytes（ hiPSC-CMs） and induce it to differentiate into pacemaker-like cells. Methods：Human iPSC-CMs were obtained from Beijing Cellapy Biological Technology Co.,Ltd. After purified for 1 week,the hiPSC-CMs were transfected with Tbx18 and GFP carried by adenoviral vectors. Quantitative Real time-PCR was used to detect the expression change fold of HCN4 mRNA. Results： After transfection,the fluorescence could be seen by the fluorescence microscope,and it last for about 3 weeks. Within days of transduction with Tbx18,the cardiomyocytes in culture developed spontaneous electrical firing physiologically indistinguishable from that of sinoatrial node cells,along with morphological and epigenetic features characteristic of pacemaker cells. Conclusion： Tbx18 can be transfected into hiPSC-CMs,and it can induce human induced pluripotent stem cell-derived cardiomyocytes to differentiation into sinoatrial node-like pacemaker cells.